Effect Of GPNMB Stored In Extracellular Matrix Of The Lung On Silicosis Fibrosis

Background:Silicosis is a disease characterized by pulmonary fibrosis caused by long-term inhalation of free silica（Si O₂）.Fibroblast proliferation and abnormal deposition of Extracellular Matrix（ECM）are the main manifestations.Recent studies have found that the ECM can provide structural support for cells,can also be involved in tissue remodeling and wound repair,after the RGD sequence structure field exposure,connect and activate the integrin,and cytoskeleton tension by intracellular signaling molecules effects,induced changes in cell morphology and related signal transduction cascade,ultimately affect the related gene expression,regulate cell function,such as proliferation,migration,adhesion and so on.Existing studies have proved that it is feasible to obtain acellular ECM from natural lung and transplant it into cells,and proteomics based on mass spectrometry has become a method to study ECM in tissues and organs.Based on these methods,researchers found that in the process of pulmonary fibrosis,fibroblasts activate into myofibroblasts and secrete a large number of ECM-related proteins such as collagen and fibronectin,resulting in fibrotic changes in ECM.In addition,studies have found that in Idiopathic pulmonary fibrosis（IPF）,due to the interaction between cells and ECM,the up-regulated pro-fibrosis factor TGF-β,or the increased content of certain proteins,such as collagen and fibronectin,in pulmonary fibrosis like ECM,In addition,increased ECM surface roughness or stiffness can promote fibroblast activation,which in turn accelerates the development of fibrosis.However,it remains unknown whether this phenomenon is also present in silicosis models.Therefore,in this topic,silicosis in mice model was established to observe fibrotic ECM influence on pulmonary fibroblast function,joint ECM proteomics,single cell RNA sequencing and space transcriptome sequencing analysis,explore the silicosis fibrosis sample,possible mechanisms of ECM affect lung fibroblast function,for target treatment of silicosis fibrosis is the ECM to provide new theoretical basis.Methods:1)Effect of fibrotic ECM on lung fibroblasts function in silicosis.a)Silica suspension（50mg/m L,per 100μL）was applied to the trachea of mice to establish a silicosis model,isolated lung tissue and prepared acellular ECM.The quality of acellular ECM and the difference of ECM was evaluated by H&E,Masson and immunofluorescence staining.b)Mice lung fibroblasts（MLG）were used to recellularize the acellular matrix,the biocompatibility of ECM were evaluated by H&E,Masson,immunofluorescence staining,Western blottingting（WB）and q RT-PCR.c)After recellularized MLG,the changes of cell function were observed by CCK-8 assay,cell recovery count,and 3D collagen cell migration assay.2)Screening and validation of key proteins in pulmonary fibrotic ECM.a)ECM proteomics analysis was performed to compare the difference in protein composition of lung ECM between the two groups,and screened a key protein-Glycoprotein nonmetastatic melanoma（GPNMB）.b)WB and immunohistochemical staining were used to verify GPNMB expression;c)Mouse recombinant protein and mouse neutralizing antibody were used to verify the effect of GPNMB on cell function.3)Cell source analysis of GPNMB on lung fibrotic ECM.a)The cell origin of the protein was analyzed by single cell m RNA sequencing and spatial transcriptome sequencing;b)Clodronte liposomes（5mg/m L,per 100μL）were injected into the tail vein of silicosis mice,to explore the fibrosis degree of pulmonary ECM and the expression of GPNMB,and the effects of these changes on lung fibroblast function;c)Database analysis was conducted to explore the possible mechanism of the effect of GPNMB on lung fibroblast function on pulmonary fibrotic ECM.Results:1)The pulmonary ECM of silicosis mice showes fibrotic changes,fibrotic ECM can affect the activation,proliferation and migration of pulmonary fibroblasts.a)56 days after a single trachea infusion of Si O₂ suspension in mice,high-density shadow appeared in the lungs,decreased lung function related indicators and lung tissue structure disorder,indicating that the silicosis model was successfully established.b)The lung ECM was successfully decellularized with ionic detergent,salt solution and DNAase solution,and the lung acellular matrix was successfully transplanted;c)Lung ECM tissue structure disorder,collagen deposition,ECM-related proteins such as Collagens,Fibronectins（FN）increased content,fibrotic changes;d)Fibrotic ECM in silicosis mice can improve the activity of lung fibroblasts and promote cell proliferation,migration and activation.2)The key protein GPNMB stores on fibrotic ECM and involves in the change of lung fibroblastic function.a)Glycoprotein nonmetastatic melanoma（GPNMB）is the key protein affecting the function of lung fibrotic ECM in silicosis mice.b)Increased GPNMB protein content in lung ECM protein;c)Fibroblasts were stimulated with recombinant mouse GPNMB protein（0.1μg/m L）,and cell viability,proliferation,migration and activation were all promoted.d)After ECM was pretreated with mouse GPNMB neutralizing antibody（0.5μg/m L）,the promoting effect of pulmonary fibrotic ECM on proliferation and migration of lung fibroblasts was attenuated.3)Macrophages are one of the important cell sources of GPNMB in lung ECM of silicosis mice.a)Single-cell RNA sequencing and spatial transcriptome sequencing indicated that one of the main cell sources of GPNMB was macrophages,and gpnmb m RNA and macrophages were mainly concentrated in the fibrosis lesions at the late stage of fibrosis;b)After 56 days of injection of Clodronte liposomes into the tail vein of silicosis mice,the degree of lung fibrosis was reduced,the fibrotic changes of lung ECM were attenuated,and the content of GPNMB protein in lung tissue and lung ECM was reduced;c)After the lung fibroblasts were transplanted into macrophages to remove the acellular ECM from the lung of mice,the cell viability was reduced,and the promoting effect on cell proliferation and migration was reduced;d)CD44 may be the surface receptor of GPNMB in lung fibroblasts,affecting cell function and participating in the progression of pulmonary fibrosis.Conclusion:In the development of silicosis,normal lung ECM develops into fibrotic ECM,and macrophages activate to produce GPNMB,secretes and stores in fibrotic ECM,recognize the pulmonary fibroblastic receptor-CD44,and then change the function of cells,promoting pulmonary fibrosis.These results suggest that GPNMB in fibrotic ECM may be a new direction to delay or even reverse silicosis fibrosis.